phytopathogenic burkholderia species in bulb plants in bulgaria

2477Biotechnol. & Biotechnol. eq. 25/2011/3

Article DOi: 10.5504/bbeq.2011.0054 b&e

biODiVerSitY AND ecOSYSteMS

Biotechnol. & Biotechnol. eq. 2011, 25(3), 2477-2483Keywords: Burkholderia cepacia, Burkholderia gladioli, phenotypic diversity, antagonistic properties

Introductionthe genus Burkholderia represents a group of closely related bacteria with a great adaptability and metabolic potential. the genus comprises more than 40 species, which occupy a wide range of ecological niches. it includes soil, water and rhizosphere saprophytes, plant, animal and human pathogens, and endosymbionts. their biological and metabolic properties can be exploited for biocontrol, bioremediation and plant growth promotion. Some of them are universal contaminants of cosmetic and pharmaceutical solutions. the most important Burkholderia species in terms of pathogenic potential are Burkholderia mallei and Burkholderia pseudomallei and the Burkholderia cepacia complex. in humans B. cepacia complex bacteria have been associated with a wide variety of infections, most often in patients with cystic fibrosis. The Burkholderia group of plant pathogens presently includes Burkholderia andropogonis, Burkholderia caryophylli, B. cepacia, Burkholderia gladioli, Burkholderia glumae and Burkholderia plantarii, which are etiological agents of diseases for a variety of plants, and cause symptoms such as wilt, rot, blight, or cancer. B. cepacia is the causal agent of “sour skin”, which is primarily a disease of onion (7), and has been reported from onion-growing areas all over the world (16). B. gladioli was originally described as a phytopathogen on Gladiolus species. however, other hosts include onion (“slippery skin”), iris and freesia (25). B. gladioli has also been reported as a pathogen of Dendrobium orchids (26) and rice plants (8). the disease “slippery skin” was first described in Bulgaria as “mealy soft rot of onion” by Vitanov (27-30). this study included symptoms descriptions, biochemical characterization of the

causal agent, and measures for disease control. especially great damages occurred in 1964, when 50% of the onion crops in Veliko tarnovo valley were destroyed. Since then the phytopathogenic bacteria of the genus Burkholderia have not been studied in Bulgaria.

The aim of this study was the molecular identification and phenotypic diversity among Burkhoderia species pathogenic to onion in Bulgaria.

Materials and MethodsPlant samples and bacterial strainsthe strains included in this study originated from infected plant material with symptoms of bacteriosis. Plant samples were collected in aseptic conditions from diseased scales of onion bulbs (Allium cepa) according to the methodology described by Klement, 1990 (18). Bacterial strains were obtained as distinct colonies on King’s medium B after cultivation at 28°c for 48-72 h. Selection of strains was made on the basis of screening for genus differentiating properties: oxidase and catalase activity, Gram-reaction, motility and cell morphology and after purification procedures. Pathogenicity of the strains was confirmed by an artificial inoculation of onion bulb scales and whole onion bulbs. Initial species identification of the isolates was carried out by the miniaturized identification system BioloGtM (BioloGtM, cA, USA) (6).

two type strains - Burkholderia cepacia nBiMcc 8566 (lMG 1222, Atcc 25416), and Burkholderia gladioli pv. gladioli nBiMcc 8569 (Atcc 10248, lMG 2216) and three clinical isolates of Burkholderia cepacia (nBiMcc 3933, nBiMcc 3934, nBiMcc 3935) were used as controls.

the following microorganisms were used as tests in examination of the interspecies interactions: Bacillus subtilis nBiMcc 2353 and nBiMcc 1709, E. amylovora nBiMcc

PHYTOPATHOGENIC BURKHOLDERIA SPECIESIN BULB PLANTS IN BULGARIA

Mariya Stoyanova1, Yoana Kizheva2, Valentina chipeva2, nevena Bogatzevska1, Penka Moncheva2

1Plant Protection institute, Kostinbrod, Bulgaria2Sofia University ”St. Kliment Ohridski”, Faculty of Biology, Sofia, Bulgariacorrespondence to: P. MonchevaE-mail: [email protected]

ABSTRACTThe identification of 22 isolates as Burkholderia cepacia and B. gladioli was confirmed by PCR amplification with species-specific primers CMG 16-1, G 16-2, CMG 23-1, and CM 23-2. The strains were characterized phenotypically by BIOLOG metabolic profile, antibiotic susceptibility, and interactions with other microorganisms. A greater metabolic diversity among B. gladioli strains than among B. cepacia strains was revealed. The phytopathogenic and the clinic isolates of B. cepacia were clearly distinguished in the studied properties. The phytopathogenic isolates of B. cepacia differed from the clinic strains in their greater metabolic abilities, significantly lower susceptibility to the tested antibiotics, and ability to synthesize antimicrobial substances against Gram-positive and Gram-negative bacteria as well as fungi.

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8484, 8475, 8477, and 8483, Micrococcus luteus nBiMcc 159, Pseudomonas aeruginosa nBiMcc 1390, Staphylococcus aureus nBiMcc 3359, Fusarium graminearum nBiMcc 2293, Fusarium moniliforme nBiMcc 394, Fusarium oxysporum nBiMcc 124, Saccharomyces cerevisiae nBiMcc 537, Candida albicans nBiMcc 72 and nBiMcc 74, Clavibacter michiganensis subsp. michiganensis, Serratia sp., Stenotrophomonas sp., Xanthomonas axonopodis pv. phaseoli, Xanthomonas campestris pv. campestris, Xanthamonas vesicatoria pv. tomato race t1, Xanthomonas axonopodis pv. glycines. the last seven test-bacteria were kindly provided by Prof. Bogatzevska (Plant Protection institute, Kostinbrod).

Molecular identificationDnA extraction was performed after cultivation of the strains in luria-Bertrani Broth at 28°c, 200 rpm for 24 h. cell density of the suspensions was measured on a spectrophotometer (Genequant Pro, Amersham technologies, UK) at 600 nm. A quantity of each strain suspension corresponding to 2x109cells/ml was taken for centrifugation and further processing. DnA extraction was performed by Dneasy Blood & tissue Purification Kit (QIAGEN GmbH, Germany) according to the manufacturer’s instructions. control of yield and purity of obtained DnA was performed by measuring on a spectrophotometer (Genequant Pro, Amersham technologies, UK) at 230 nm, 260 nm, 280 nm, and 320 nm. Final volumes with DNA concentration of 70-100 μg/ml were stored at -20°C until further use.

PCR amplifications were carried out in a multiplex PCR with species-specific primers purchased from LKB Vertriebs Gmbh (Austria): cMG16-1 (5’AGA Gtt tGA tcM tGG ctc AG3’), G 16-2 (5’cGA AGG AtA ttA Gcc ctc 3’), cMG 23-1 (5’AtA Gct GGt tct ctc cGA A3’), and cM 23-2 (5’ctc tcc tAc cAt GcG (ct)G c3’) (5). each PcR reaction was carried out in a 25 μl final volume containing 2.5 μl reaction buffer, 0.9 μl MgCl2 (50 mM), 0.5 μl of each nucleotide primer (100 pM/μl), 0.187 μl dNTPs, 0.08 μl STS-Taq polymerase, and 1 μl DNA, under the following reaction conditions: initial denaturation at 95°c for 5 min, 25 cycles of 95°C for 5 min, 57°C for 30 s, and 72°C for 45 s, and a final elongation step at 72°c for 5 min.

the PcR products were separated electrophoretically in 1% agarose gel in 1xtBe buffer, (1 h 30 min at 100 V), stained with ethidium bromide, and visualized at 302 nm with a UV transiluminator system GenoPlex (VWR international, llc, USA). PcR products on the gel were analyzed by the included software.

Phenotypic characterizationthe morphology of the colonies was observed on nutrient agar containing: 10 g/l meat extract, 10 g/l peptone and 5 g/l nacl, after cultivation at 28°c for 24 to 48 hours.

the biochemical characterization of the isolates was done by the miniaturized system BioloG (BioloG inc., hayward, cA, USA). Utilization of 95 carbon substrates was examined

by BioloG Gn2 microplates, following the manufacturer’s instructions.

Cluster analysisthe data received from BioloGtM Gn microplates was analyzed by cluster analysis according to Ward’s method and matrix of similarity on the basis of squared euclidean distance.

Antibiotic susceptibilityIn vitro susceptibility to 14 antibiotics from different chemical groups was tested by the Kirby-Bauer technique (4) using the following sensitivity discs: penicillin NCIPD (6 μg/disk), piperacillin NCIPD (100 μg/disk), ceftazidim NCIPD (30 μg/disk), amoxicillin NCIPD (25 μg/disk), augmentin Oxoid (20 μg/disk/10 μg/disk), gentamycin NCIPD (10 μg/disk), kanamycin NCIPD (30 μg/disk), chloramphenicol NCIPD (30 μg/disk), doxycyclin NCIPD (30 μg/disk), trimetoprim Oxoid (5 μg/disk), sulphometoxazol-trimetoprim NCIPD (23.75 μg/disk/1.25 μg/disk), nalidixic acid NCIPD (10 μg/disk), and pefloxacin Oxoid (5 μg/disk), rifampicin Oxoid (5 μg/disk). Bacterial suspensions of about 109 cells/ml were prepared in physiological saline from 24-h-old nutrient agar cultures and then plated on Petri dishes containing the same medium. Antibiotic disks were placed on the surface of the inoculated medium. After incubation at 28°c for 24 h the diameter of the inhibition zones was measured.

Interactionsthe antimicrobial activity of the isolates was examined by the well diffusion method. the isolates were cultivated in nutrient broth at 28°c for 24 h to a density of approximately 107 cells/ml. Then cultures were filtrated through filters with 0.22 μm pores to remove the cells. the target test-microorganisms were plated on an appropriate agar media for each of them. the cell density standardized inoculum of 0.1 ml was used. A quantity of 50 μl of the cell-free extract was dropped in each well. After incubation at 28°c for 24-36 h the presence of inhibition zones was recorded.

Results and DiscussionPlant samples and bacterial strainsthe inspected plant samples (bulbs) had soft beige-yellow diseased scales with а weak to strong smell of boiled onion with a sour tinge.

the isolated bacterial strains gave round shaped beige-yellowish colonies on King’s B medium, raised, with a straight edge, approximately 2-2.5 mm/diameter for three-day cultivation. the surface of the colonies was either smooth (the type strain of B. cepacia and eight isolates) or rough and wrinkled for the rest isolates and the type strains of B. gladioli pv. gladioli.

After isolation and purification procedures twenty-two bacterial strains were obtained, 13 of which produced a dark brown diffusible pigment. All of them induced hR in tobacco leaves. Pathogenic on onion scales were also 21 strains. Up to three days after inoculation, the infected tissues became


watery and soft, with a color varying from yellow to brown, and a weak to medium smell of boiled onion with a sour tinge, while no changes were observed in the non-inoculated controls. three of the strains caused reddish pigmentation of the scales. the infected bulbs germinated with thin slightly chlorotic malformed leaves up to the 15th day after inoculation. inside, the bulbs showed clear symptoms of bacteriosis with soft, water-soaked, beige-yellow to brown colored tissues with а sour smell. The type cultures of B. cepacia and B. gladioli pv. gladioli provoked the same reactions which indicated the similarity between the isolates and type cultures.

Molecular identificationThe isolates were identified by BIOLOG as B. cepacia, Burkholderia pyrrocinia, and B. gladioli (6). This identification was confirmed by PCR amplification with species-specific primers for B. cepacia and B. gladioli. All of the tested strains gave a positive result for the presence of the specific gene regions in the area coding 16S and 23S ribosomal RnA (rRnA) with primers cMG 16-1, G 16-2, cMG 23-1, and cM 23-2: 13 strains gave amplification typical for B. gladioli and 8 strains, amplification that is characteristic for B. cepacia, including the three strains previously identified as B. pyrrocinia by the BioloGtM system (6) (Fig. 1 and Fig. 2). it is notable that the strains identified by BIOLOG as B. pyrrocinia generated the same amplification product as B. cepacia.

Fig. 1. Multiplex PCR amplification of Burkholderia sp. with the primers cMG 16-1, G 16-2, cMG 23-1, and cM 23-2. the number of each lane corresponds to the designation of the strain analyzed; M: DnA marker 100 bp (novagen, eMD chemicals inc., USA).

Fig. 2. Multiplex PCR amplification of Burkholderia sp. with the primers cMG 16-1, G 16-2, cMG 23-1, and cM 23-2. the number of each lane corresponds to the designation of the strain analyzed; M: DnA marker 100 bp (novagen, eMD chemicals inc., USA).

the species from B. cepacia complex are phenotypically and genotypically very close and it is difficult to distinguish them (21). B. pyrrocinia is a member of B. cepacia complex (genomovar iX). there can be found a high level of 16S rDnA sequence similarity (>97.7%) between these bacteria (24). the coding region for 23S rRnA is highly conservative as well as the region for 16S rRnA, which can explain the presence of an amplification product both for B. cepacia and B. pyrrocinia with the used primers. Similar results were obtained with E.

amylovora and Erwinia pyrifoliae by Atanasova et al. (2, 3) after amplification with species-specific primers for the ams region of E. amylovora.

At this stage of our study, using only two pairs of primers, we could not reliably assume that we had found B. pyrrocinia in the onion bulbs. however, the symptoms on onion after artificial inoculation caused by the strains identified by BioloG as B. pyrrocinia were different from those caused by the B. cepacia strains, namely – the color of the infected tissues was reddish and not yellow. Until now, there is no data about the synthesis by B. pyrrocinia of specific pigments, only antibiotics have been purified – bromo-derivatives of pyrrolnitrin, named bromonitrin A, B, and c (1, 12). the pyrrolnitrin crystal is pale yellow in color but when expressed in Pseudomonas fluorescens, pyrrolnitrin B is red – variations due to its chemical and stereo-structure (9, 31). Although such investigations were not subject to this study, the synthesis of such an antibiotic is a possible explanation. Based on the summoned knowledge for the species B. pyrrocinia the reasons for the pigmentation of the plant tissues remain obscure.

Macromorphologythe isolates had two types of colonies that showed species dependency. The first type consisted of beige, circular, raised, gleamy, heterogeneous colonies with an entire edge. the old colonies were with wrinkled surface (B. gladioli nBMicc 8569 and all isolates identified as B. gladioli). the second type of colonies were yellow, circular, convex, gleamy, and homogeneous with an entire edge (B. cepacia nBMicc 8566 and all isolates identified as B. cepacia and B. pyrrocinia). Variation in the colony morphology did not occur within any of the species (Fig. 3).

Fig. 3. Morphological types of colonies

Metabolic profile and cluster analysisthe data obtained by the BioloG system were used to assess the intraspecies metabolic diversity. they were subjected to cluster analyses which defined four groups at 80% similarity (Fig. 4). this grouping was on the basis of the same reaction of the strains to 27 substrates included in the BioloG system and their diverse reaction to the rest 68 substrates.


TABLE 1Biolog Gn substrates differentially used by the strains identified as B. cepacia and associated to the groups defined by 80% similarity

SubstrateCluster

A BDextrin - (66.7)Glycogen - (66.7)n-Acetyl-D-galactosamine - (22.2)D-cellobiose - (77.7)i-erythritol - (11.1)Gentiobiose - +β-Methyl-D-Glucoside - (12.5)D-Psicose - (11.1)D-trehalose 33.3; (66.7) 77.7; (22.2)Xylitol + -Acetic acid (100) 88.9; (11.1)D-Galactonic Acid lactone + 33.3; (55.6)D-Glucosaminic Acid - 33.3; (55.6)α-Hydroxybutiric Acid + 44.4; (55.6)γ-Hydroxybutiric Acid - (22.2)p-hydroxy Phenylacetic Acid (66.7) 44.4; (55.6)itaconic acid - (11.1)α-Ketobutyric acid (100) 44.4; (55.6)α-Ketoglutaric acid - (11.1)α-Ketovaleric acid - (66.7)Sebacic acid + 88.9; (11.1)Succinamic acid - 55.5; (11.1)Glucoronamidе (33.3) 11.1; (77.8)l-Alaninamide - 44.4; (11.1)l-Alanyl-glycine (66.7) 22.2; (66.7)l-leucine - (88.8)l-ornithine - (88.8)l-Phenylalanine (100) 55.6; (44.4)l-Serine 66.7; (33.3) 66.7; (33.3)l-threonine - 55.6; (44.4)D,l-carnitine - 66.7; (33.3)Uridine - (11.1)thymidine - 11.1; (88.9)Phenyl-ethyl-amine (100) 55.6; (44.4)Putrescine (100) +2-Aminoethanol - +2,3-Butanediol - 33.3; (44.4)Glycerol - 33.3; (11.1)D,L-α-Glycerol Phosphate - +α-D-Glucose-1-Phosphate - 22.2; (22.2)+: all the strains in the group were positive; -: all the strains in the group were negative; number: percentage of positive strains in the group; number in brackets: percentage of weakly positive strains in the group

The strains identified as B. cepaciа were grouped in two clusters. The first cluster A included all the clinic strains of B. cepacia, which were used in this study as controls. no great diversity could be found among the strains in this cluster. They differed in the ability to assimilate five substrates. The second cluster B grouped five phytopathogenic strains which were identified as B. cepacia, as well as the type culture. the cluster included also the three strains identified by BIOLOG as B. pyrrocinia which separated at only 92 to 97% similarity and had not been distinguished from B. cepacia strains by the used molecular method. this cluster was formed on the basis of the common reaction of all the strains to 60 substrates. the main variations among the isolates were in the different levels of assimilation of some substrates (Table 1).

Fig. 4. Dendrogram showing BioloG clusters of Burkholderia strains. the numbers correspond to the designation of the strains analyzed; *: the nBiMcc numbers of type cultures and reference strains

the differences between the two clusters were based on their reaction to 40 substrates (Table 1). Significant differences could be noted between the phytopathogenic and clinic strains, which had only 52% similarity. twenty six substrates which were assimilated from the phytopathogenic strains were not utilized by the clinic ones. only three substrates were assimilated by all of the phytopathogenic isolates, but not of the clinic ones, and xylitol was the substrate which was assimilated by all of the clinic strains, but not by the phytopathogens (Table 1). hence, it may be concluded that phenotypic diversity between the phytopathogenic and clinic populations of B. cepacia occurred, which may be due to differences in their habitats and the available substrates. the phytopathogenic population clearly possessed greater metabolic abilities than the clinic one, which probably ensures its survival in the comparatively unstable environment.


TABLE 2Biolog Gn substrates differentially used by the strains identified as B. gladioli and associated to the groups defined by 80 % similarity

SubstrateCluster

C DDextrin - 12.5; (12.5)Glycogen 50; (50) 12.5; (25)n-Acetyl-D-galactosamine + 62.5; (37.5)D-cellobiose + (12.5)α-D-Glucose (83.3) +lactulose 33.3; (16.7) -Maltose 33.3; (16.7) -D-Mannose 66.7; (33.3) +D-Melibiose 33.3; (50) (12.5)β-Methyl-D-Glucoside 16.7; (50) (12.5)D-Psicose (83.3) (75)D-Raffinose 50; (50) (12.5)Sucrose 16.7; (50) -turanose 50 -Pyruvic Acid Methyl ester 16.7; (83.3) 75; (25)Acetic acid 33.3; (66.7) 50; (50)cis-Aconitic acid 66.7; (33.3) +citric acid 66.7; (33.3) +Formic acid 66.7; (33.3) +D-Glucoronic acid - (12.5)itaconic acid - (50)α-Ketobutyric acid 83.3; (16.7) 87.5; (12.5)α-Ketoglutaric acid 66.7; (33.3) 25; (25)α-Ketovaleric acid 33.5; (50) 25; (25)D,l-lactic acid 66.7; (33.3) 50; (50)Malonic acid 66.7; (33.3) +Propionic acid 66.7; (33.3) 75; (25)Sebacic acid 16.7 (83.3) 37.5; (62.5)Succinic acid 16.7 (83.3) +Bromosuccinic acid + 87.5; (12.5)Succinamic acid (50) 12.5; (12.5)Glucoronamidе 16.7 (83.3) 25; (25)l-Alaninamide (16.7) 25; (25)l-Alanine + 87.5; (12.5)l-Alanyl-glycine 33.3; (66.7) 75; (25)l-histidine 16.7; (83.3) 75; (25)hydroxy-l-proline + 87.5; (12.5)l-leucine (16.7) 12.5; (12.5)l-ornithine (16.7) 12.5l-Phenylalanine + 62.5; (37.5)l-Proline + 87.5; (12.5)l-Pyroglutamic Acid 83.3 87.5l-threonine + 37.5; (62.5)

D,l-carnitine (66.7) (75)Urocanic Acid 16.7; (83.3) 12.5; (25)inosine 16.7; (83.3) (37.5)Uridine (16.7) -thymidine (16.7) 12.5; (12.5)Phenyl-ethyl-amine - (25)Putrescine 33.3 -Glycerol 16.7; (83.3) (37.5)D,L-α-Glycerol Phosphate 33.3; (50) 12.5; (50)α-D-Glucose-1-Phosphate (16.7) (12.5)D-Glucose-6-Phosphate 83.3 62.5; (37.5)+: all the strains in the group were positive; -: all the strains in the group were negative; number: percentage of positive strains in the group; number in brackets: percentage of weakly positive strains in the group

The third cluster C consisted of six strains identified as B. gladioli and the variations in the group were much more than those between the members of the second cluster B. the fourth group D consisted of eight strains also identified as B. gladioli, including the type culture. A common characteristic of all the B. gladioli strains was the reaction to 41 substrates. intraspecies diversity existed in relation to their ability to utilize 56.8% of the substrates (Table 2). Differences were in the level of the positive reaction to one or another substrate. Greater differences were recorded only to seven substrates – dextrin, lactulose, maltose, D-melibiose, turanose, sucrose, and putrescin. the utilization of the rest of the substrates was weaker or stronger.

Antibiotic resistancethe strains’ susceptibility to 14 antibiotics from different chemical groups was tested. it was obtained that all of the strains are resistant to penicillin, ceftazidim, amoxicillin, augmentin (with the exception of the type culture B. gladioli pv. gladioli), and rifampicin, and susceptible to piperacillin, trimetoprim, sulphometoxazol-trimetoprim, and nalidixic acid. the strains showed different reaction to gentamicin, kanamycin, chloramphenicol, doxycyclin, and pefloxacin (Table 3). The diversity among the strains identified as B. gladioli was revealed to kanamycin, chloramphenicol and pefloxacin. The phytopathogenic B. cepacia strains showed different reaction to chloramphenicol, doxycyclin, and pefloxacin. The clinic B. cepacia strains greatly differed from the phytopathogenic ones with their strong sensitivity to the five antibiotics. The data in the published literature is predominantly about the antibiotic susceptibility of clinic isolates. it was established that B. gladioli was sensitive to chinolones, aminoglycosides and amoxycylcin/clavulanic acid. the sensitivity to ceftazidim and piperacilin varied (10). About 95% of the clinic isolates of B. cepacia were susceptible to sulphometoxazol/trimetoprim, 85% to chloramphenicol, 80% to piperacillin, and 65% to doxycyclin and ceftazidim (32). Some of our results confirmed the data obtained by other authors. however, the data obtained by us revealed the diversity between the strains of one and the same species.


TABLE 3Differences in the reaction to the antibiotics

Antibiotics Percentage of the strainsB. gladioli B. cepacia (phytopathogenic) B. cepacia (clinic)R i S R i S R i S

Gentamicin 0 0 100 100 0 0 0 0 100Kanamycin 0 7.1 92.9 100 0 0 0 0 100chloramphenicol 14.3 71.4 14.3 0 11.1 88.9 0 0 100Doxycyclin 0 0 100 11.1 55.6 33.3 0 0 100Pefloxacin 0 50 50 44.4 55.5 0 0 0 100R: resistant; i: intermediate resistance; S: sensitive

TABLE 4Antagonistic properties of the strains to test microorganisms

Test microorganisms Strains from the genus Burkholderia22B 23B 24B 25B 26B 27B 28B 29B 8566 3933 3934 3935 8569 16B 18B 21B 31B 33B 35B

B. subtilis nBiMcc 1709 + - + + + + + + + - - - - - - - - - -B. subtilis nBiMcc 2353 + - - + - - + + + - - - - - - - - - -C. albicans nBiMcc 72 + - - - - - + - - - - - - - - - - - -C. albicans nBiMcc 74 + - - - - - - - - - - - - - - - - - -C. michiganensis (Germany) - - - + - - - - - - - - - - - - - - -C. michiganensis (Bulgaria) + - - + - - + + + - - - - - - - - - -E. amylovora nBiMcc 8475 + + + + + + + + + - - - - - - - - - -E. amylovora nBiMcc 8477 + + + + + + + + + - - - - - - - - - -E. amylovora nBiMcc 8484 + - + + + + + + + - - - - - - - - - -E. amylovora nBiMcc 8483 + - - + - - + + + - - - - - - - - - -M. luteus nBiMcc 159 - - - + - - - + + - - - + + - - - - +Serratia sp. (Bulgaria) + + - + + + + + + - - - - - - - - - -Stenotrophomonas sp. (Bulgaria) + + + + + + + + + - - - - - - - - - -S. aureus nBiMcc 3359 + + + - + + + + + - - - - - - + - - -X. axonopodis pv. phaseoli (Bulgaria) + + - + + - - + + - - - - - - - - - -X. axonopodis pv. glycines (Bulgaria) - - - + - - - + + - - - - - - - - - -X. campestris pv. campestris (Bulgaria) + + + + + + + + + - - - - - - - - - -

X. vesicatoria pv. tomato (Bulgaria) - - - + - - - + + - - - - - - - - - -F. graminearum nBiMcc 2293 - - + - - + - - - + + + - - + - - + -F. moniliforme nBiMcc 394 - - - - - - - - + - - - - - - - + + -F. oxysporum nBiMcc 124 - - - - - - - - + - - - - - - - - + -+: growth inhibition-: lack of growth inhibition

Interactionsthe diversity in the interactions between B. cepacia strains (including the clinic isolates), B. gladioli strains, and the test microorganisms was studied (Table 4). All of the tested strains had no antagonistic effect against P. aeruginosa or S. cerevisiae. only a few strains had an effect against C. albicans, F. moniliforme, F. oxysporum, and F. graminearum. B. cepacia phytopathogenic strains were more effective against bacteria than against fungi. B. cepacia was most effective against Stenotrophomonas sp. (100%), X. campestris pv. campestris (100%), Serratia sp.(89%), S. aureus (89%), E. amylovora (86%), and B. subtilis (72%), comparatively effective against X. axonopodis (67%), and weakly effective against C. albicans (17%), C. michiganensis (33%), M. luteus (33%), X. axonopodis pv. glycines (33%), X. vesicatoria pv.

tomato (33%). the largest antagonistic spectrum possessed strains 22B, 25B, 29B, as well as the type strain against 16, 17, and 18 test-microorganisms respectively (Table 4).

• B. cepacia did not inhibit the growth of B. gladioli strains.

• B. cepacia clinic isolates lacked the antagonistic properties of the phytopatogenic strains showing activity only against F. graminearum.

compared to B. cepacia phytopathogenic strains, the tested B. gladioli isolates had significantly lower antagonistic activity, which was more like the activity of B. cepacia clinic strains. however, this antagonistic effect was revealed to different test organisms. Unlike all B. cepacia isolates, there could be found two B. gladioli isolates with activity against F. moniliforme


and F. oxysporum. B. gladioli strains showed activity only against Gram-positive bacteria (M. luteus and S. aureus) and fungi (Table 4). Many Burkholderia species produce antimicrobial substances (B. cepacia, B. gladioli pv. gladioli, B. caryophylli, B. glumae, B. plantari) with antibacterial and antifungal properties. Various strains of B. cepacia have been reported to produce a large variety of antifungal compounds such as cepacin (22), altericidines (17), pyrrolnitrin (13, 14, 15), cepacidines (19, 20) and siderophores (23) that are iron-chelating compounds implicated in antibiosis against plant pathogens. it was suggested that some of these substances may contribute to the competitive survival of Burkholderia species in the soil (11). Our results confirmed the ability of Burkholderia species to produce antimicrobial substances and demonstrated its strain specificity.

Conclusionson the basis of the metabolic activity of the strains according to their BIOLOG profiles, susceptibility to antibiotics, and the interactions with other bacteria and fungi the phenotypic intraspecies diversity among the strains was revealed. the phytopathogenic population of B. cepacia differs greatly from the clinical strains, which may be due to the specificity of their habitats. This paper presents the first phenotypic characterization of phytopathogenic B. cepacia and B. gladioli strains in Bulgaria.

Acknowledgementsthe investigation was supported by the national Science Fund of Bulgaria (project ВУ-Б-206/06).

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